Botulinum neurotoxins (BoNT) are the most toxic substances known to humans causing respiratory failure upon poisoning. BoNT's lethality and ease of production has led to a category "A" bioterrorism agent designation for BoNT by the Department of Defense (DoD). Developing effective, post-exposure antagonists to BoNT is a top priority of the DoD. Despite their lethality, BoNTs have cosmetic and pharmaceutical applications and are currently FDA-approved for the treatment of glabellar lines (wrinkles), cervical dystonia, blepharospasm, cranial nerve VII disorders, and cosmoses. BoNT provides relief of muscle tension by silencing neurons that cause muscle contraction. For many disorders, BoNT-based treatments provide significant and long lasting reductions in pain. BoNT's exquisite specificity for neurons and long time of action make it a lead candidate for the treatment of neurological and muscle disorders where conventional treatments have failed. Development of new BoNT-based therapies is hindered by the lack of assays to measure BoNT activity. Currently, the only widely accepted method of measuring BoNT activity involves injecting BoNT into mice and counting the number of mice that die after one or more days. Because this process takes days to complete, requires special facilities, and is extremely low-throughput, it makes antagonist screening or development of new BoNT-derived therapeutics prohibitively expensive and time-consuming. The overall objective of this Phase I proposal is to develop a unique technology for measuring BoNT activity in living cells. Because BoNT has three activities-cell receptor binding and uptake, vesicle translocation, and target cleavage-a cell-based assay is required to effectively measure BoNT activity. BioSentinel proposes to develop a cell-based assay that measures BoNT activity by using Forster resonance energy transfer (FRET) to monitor BoNT cleavage of SNARE proteins, the target of BoNT. BioSentinel will generate FRET-based reporters for measuring BoNT protease activity, identify cell lines that are highly sensitive to BoNT, generate stable cell lines that express the reporters, and investigate high- throughput instruments for monitoring those reporters in vivo. BioSentinel proposes to develop the assay into a high-throughput platform that will enable researchers and pharmaceutical companies to rapidly quantify BoNT preparations, perform quality control measures, screen for new BoNT-based therapies, and screen for BoNT antagonists. Because of increased pressure for animal-free, high-throughput assays, a cell-based assay for BoNT detection is widely desired by researchers and industry, and would open a gateway to development of new BoNT-based therapies. Public Health Relevance: Botulinum neurotoxins are extremely lethal bacterial toxins that attack the nervous system and are considered a significant bioterrorism threat. Botulinum neurotoxins are also widely used for cosmetic and pharmaceutical applications providing relief of muscle contraction and pain. BioSentinel proposes to develop a high-throughput assay for measuring botulinum neurotoxin activity in living cells, providing a much needed platform for screening antagonists to BoNT and developing new BoNT-based therapies.